A number of studies now implicate an increase in reactive oxygen species generation (ROS) in the pathogenesis of many cardiovascular diseases and this increase is believed to play a major role in the development of endothelial dysfunction. A major consequence of endothelial dysfunction is a decrease in bio-available nitric oxide (NO). The overall goals of our studies are to understand how reactive oxygen species (ROS) modulate endothelial nitric oxide synthase (eNOS) activity. Our recent studies have identified a novel reversible mechanism by which eNOS can be inhibited. We have found that hydrogen peroxide (H202) can induce a disruption of the eNOS dimer. However, recent published studies indicate that H202 can also be involved in activating the eNOS enzyme. In this proposal, we will test the hypothesis that the underlying mechanism for this dichotomous effect of H202 on eNOS activity is due to the endothelial cells ability to metabolize H202. In vivo, using primary cultures of pulmonary and aortic endothelial cells isolated from various gestational ages, we will determine whether there are developmental or vascular bed specific effects of H202 on eNOS activity. We will then follow up these studies by elucidating the mechanisms for the stimulatory and inhibitory effects of H202 on eNOS. Our recent studies indicate that the activation of eNOS by H202 appears to be linked to a c-Src mediated phosphorylation of HS90 and caveolin 1. The roles of these proteins in Enos activation by H202 will be elucidated using a COS-7 cell line stably expressing eNOS as a model system. We will then determine the effect of transient transfections with various wild type, dominant negative and dominant positive expression plasmids for caveolin 1, HSP90, and c-Src on H202-mediated eNOS activation. We will then determine the mechanism mediating the dimer disruption induced by H202. We will test the hypothesis that H202-mediated oxidation of tetrahydrobiopterin is involved in eNOS dimer disruption. We will investigate this in vivo using primary cultures of endothelial cells and in vitro using recombinant human eNOS protein purified from an E.coli expression system we have developed. The information garnered from the successful completion of these studies should prove to be significant in a number of physiological processes. Clinically, these studies may have important implications in interventions directed at pathophysiological disorders in which excess ROS production is though to be involved. These include pulmonary and systemic hypertension, diabetes, and atherosclerosis.